Re: Borrelia genetics

a_weisman_at_yahoo.com
Date: 02/27/05 

Date: 27 Feb 2005 06:32:31 -0800

eugeneshapiroisapig wrote:
> Hey guys, I could spend a lot of time at the library looking this up
on
> my own, or I could save time and ask here. What I am interested in
now
> are basic facts about the molecular biology of Borrelia species other
> than Borellia burgdorferi. By which I mean hermsii, etc., you know
the
> stuff that causes relapsing fever and things like this. In
particular,
> I am interested now in the genetic organization of these bugs.
> Burgdorferi has I believe 9 circular plasmids, 12 linear plasmids,
and
> a linear chromosome. What about other borrelia? Also interested in
> worldwide distribution of hermsii. Any other information about
> comparative cell biology or evolution would also be appreciated.
Thanks.

Thought you might be interested in this too (better to read it at the
site that way you can view the tables and access references and
supplementary materials):

Comparative analysis of the Borrelia garinii genome -- Glöckner et al.
32 (20): 6038 -- Nucleic Acids Research
http://nar.oupjournals.org/cgi/content/full/32/20/6038

Nucleic Acids Research 2004 32(20):6038-6046; doi:10.1093/nar/gkh953
This Article

Published online 16 November 2004
Nucleic Acids Research, Vol. 32 No. 20 © Oxford University Press 2004;
all rights reserved

Comparative analysis of the Borrelia garinii genome
G. Glöckner*, R. Lehmann, A. Romualdi1, S. Pradella, U.
Schulte-Spechtel2, M. Schilhabel, B. Wilske2, J. Sühnel1 and M.
Platzer
Genome Analysis, Institute for Molecular Biotechnology, Beutenbergstr.
11, 07745 Jena, Germany, 1 Biocomputing Group, Institute for Molecular
Biotechnology, Beutenbergstr. 11, 07745 Jena, Germany and 2
Max-von-Pettenkofer Institut für Medizinische Mikrobiologie und
Hygiene München

* To whom correspondence should be addressed. Tel: +49 3641 656440;
Fax: +49 3641 656255; Email: gernot@imb-jena.de
Present address: S. Pradella, German Collection of Microorganisms and
Cell Cultures (DSMZ) Braunschweig, Germany
+CP000013-CP000015, AY722917-AY722953

Received September 17, 2004; Revised October 19, 2004; Accepted
November 1, 2004

DDBJ/EMBL/GenBank accession nos+

ABSTRACT

Three members of the genus Borrelia (B.burgdorferi, B.garinii,
B.afzelii) cause tick-borne borreliosis. Depending on the Borrelia
species involved, the borreliosis differs in its clinical symptoms.
Comparative genomics opens up a way to elucidate the underlying
differences in Borrelia species. We analysed a low redundancy
whole-genome shotgun (WGS) assembly of a B.garinii strain isolated from
a patient with neuroborreliosis in comparison to the B.burgdorferi
genome. This analysis reveals that most of the chromosome is conserved
(92.7% identity on DNA as well as on amino acid level) in the two
species, and no chromosomal rearrangement or larger
insertions/deletions could be observed. Furthermore, two collinear
plasmids (lp54 and cp26) seem to belong to the basic genome inventory
of Borrelia species. These three collinear parts of the Borrelia genome
encode 861 genes, which are orthologous in the two species examined.
The majority of the genetic information of the other plasmids of
B.burgdorferii is also present in B.garinii although orthology is not
easy to define due to a high redundancy of the plasmid fraction. Yet,
we did not find counterparts of the B.burgdorferi plasmids lp36 and
lp38 or their respective gene repertoire in the B.garinii genome. Thus,
phenotypic differences between the two species could be attributable to
the presence or absence of these two plasmids as well as to the
potentially positively selected genes.

INTRODUCTION

The genus Borrelia comprises 19 species of which 10 belong to the
Borrelia burgdorferi sensu lato complex (1). Only three species of this
complex cause the multisystem disorder Lyme borreliosis, B.burgdorferi
sensu stricto, B.garinii and B.afzelii. In the United States,
B.burgdorferi sensu stricto is the only causative agent for this
disease (2), whereas in Europe B.garinii as well as B.afzelii are major
contributors to the reported case numbers (3,4).

All these borreliae live in the gastro-intestinal tract of ticks
(Ixodes spec.) and are able to infect multiple hosts via tick bite. The
host range is thought to be defined by gene variations on a group of
redundant plasmids (5-7). Furthermore, the disease patterns observed
in humans are dependent on the particular Borrelia species involved.
B.garinii is primarily associated with neuroborreliosis (3), B.afzelii
with acrodermatitis chronica athrophicans (a chronic skin disease) (8),
whereas B.burgdorferi sensu stricto was found to be prevalent in Lyme
arthritis (9), which, however, was not confirmed by two other studies
(10,11).

The nuclear genomes of all B.burgdorferi sensu lato species consist of
one linear chromosome and varying amounts of several linear and
circular plasmids (12). The chromosomes are highly similar (13), but
plasmids show a wealth of diversity and can be lost during culture of
the bacteria (14,15). The genomic sequence of the B.burgdorferi B31
chromosome was determined in 1997 (16). The sequence analysis of
B.burgdorferi sensu stricto showed that the chromosome has a length of
0.94 Mb harbouring all genes for basic cellular functions. The plasmids
often share sequence motifs and segments, and seem to contain a large
number of fragmented genes, since several sequence motifs occur in
predicted non-coding as well as coding regions. This fact and the lack
of orthologs in other species made it difficult to define genes (17).
Although the plasmid fraction of the genome seems to be responsible for
host range selection and pathogenicity (6,18), large parts of it are
likely to be dispensable for viability in culture and are highly
variable, even in a single species (19).

The Borrelia species belong to the spirochetes, a group of bacteria
that have long, helically coiled cells. To date, the genomes of the
following spirochete species are known in addition to Borrelia
burgdorferi: Treponema denticola, Treponema pallidum and Leptospira
interrogans (16,20-22). Their genome sizes range from 1.6 to 4.3 Mb,
with B.burgdorferi possessing the smallest genome. The B.burgdorferi
sequence was recently used for microarray experiments to identify
similar sequences in B.hermsii (23). This analysis revealed that at
least 81% of the chromosome and 41% of the plasmid sequences of
B.burgdorferi are present in this distantly related species. Yet,
hybridization experiments can only detect highly similar sequences
shared between organisms. On the other hand, a comparative analysis of
sequences is able to detect not only low similarities that would not
give hybridization signals, but provides also access to sequence
differences of the organisms compared. Closely related species reveal
species-specific differences and evolutionary selection pressures on
genes. At the same time, a comparative sequence analysis provides the
means for a better annotation as was shown with several Saccharomyces
species (24). The comparison of more distantly related species helps to
describe core sets of proteins within a specific evolutionary branch
(25). The goal of the present comparative study was (i) to define the
orthologous gene set, which presumably is the basic set of borreliosis
causing Borreliae; (ii) to identify the stable and variable parts in
the genomes of Borrelia; (iii) to determine, which genome part is
dispensable without loosing pathogenicity; (iv) to improve the the
B.burgdorferi annotation by the B.garinii orthology information.
Furthermore, gene groups exposed to different evolutionary changes can
be defined. Since positive selection is an indicator for environmental
adaptations of the pathogen, genes evolving in such a manner are highly
suspicious to be involved in pathogenicity. Many of the B.burgdorferi
genes are so far described as only hypothetical. Thus, a comparative
analysis would also help to define true orthologous pairs and exclude
false positive predictions.

Therefore, we decided to sequence and analyse another species from the
B.burgdorferi sensu lato complex, B.garinii. Previous studies had
revealed that selected genes of both species are highly similar with
>90% sequence identity on DNA level. Moreover, there were hints that
the chromosomes of the two species are collinear (26). This relatively
high similarity enabled us to apply a low redundant sequencing
strategy. Thus, we were able to generate a complete analysis of the
chromosome and two conserved plasmids of B.garinii. The diverse plasmid
fraction of the genome could also be defined and analysed although no
clear-cut assignment to B.burgdorferi plasmids was possible due to
redundancies and rearrangements.

MATERIALS AND METHODS

The B.garinii strain PBi (OspA serotype 4), a cerebrospinal fluid (CSF)
isolate from a German patient with neuroborreliosis, was used for
sequence analysis (27). OspA-serotype 4 strains are enriched in CSF,
but they have been isolated only exceptionally from ticks (27). A low
passage of strain PBi (12th subculture) was cultured in MVP-medium as
described (28). Passage 12 is still infectious for gerbils, infectivity
was lost between passage 30 and 60. DNA was extracted using the Genomic
DNA Bufferset (Quiagen GmbH, Hilden) and Genomic tip 500/6 and 100/6
(Quiagen, GmbH, Hilden). A genomic library with a target insert size of
1.5 kb using total DNA was constructed as described previously (29).
>>From this library, 5740 clones were sequenced from both ends resulting
in an estimated coverage of the whole genome of three times. According
to the similarity of the obtained sequences to B.burgdorferi
counterparts they were binned into a plasmid and a chromosome group
using BLAST (30). The chromosomal sequences were assembled using the
assembler GAP4 (31) utilizing the finished chromosome of B.burgdorferi
(16,17) as a backbone. Using the backbone sequence as a ruler and
orientation measure, we defined primer pairs for PCR reactions to close
the gaps. For convenient primer design, we have written a Perl script
for the automatic definition of primers in a Staden package project
with the program 'primer3'
(http://www-genome.wi.mit.edu/genome_software/other/primer3.html) as
kernel (R.Lehmann, unpublished data). Nucleotide differences between
the B.garinii and the B.burgdorferi chromosome were calculated
automatically if the Phred score was 20 or better at the differing
base. Bases below this score were inspected by eye to ensure proper
difference calculation.

Initial gene finding was performed using GeneMarkS
(http://opal.biology.gatech.edu/GeneMark) (32). Orthology to
B.burgdorferi counterparts was determined by aligning the best
bidirectional hit (BBH) to each predicted protein. Only proteins
located at the same position within the different genomes and with <10%
length difference were considered as orthologs. Orphan genes of both
organisms contained no functional domains according to an InterPro
analysis (http://www.ebi.ac.uk/interpro/). Transmembrane domains were
predicted using TMHMM (http://www.cbs.dtu.dk/services/TMHMM/). The
GenBank gene descriptions of the B.burgdorferi genome (NC_001318
[GenBank] , NC_000948 [GenBank] to NC_000957 [GenBank] , NC_001849
[GenBank] to NC_001857 [GenBank] , NC_001903 [GenBank] , NC_001904
[GenBank] , NC_004971 [GenBank] ) were used for the annotation of the
corresponding B.garinii coding sequences.

To perform an independent cross-check of the reliability of this
annotation approach, all potential protein coding sequences (CDS;
potential start codon to stop codon without length threshold) of
B.garinii were used for BLASTP searches against the GenBank database.
Whenever a gene is referred to as 'hypothetical', no match in any
database could be found. A 'conserved hypothetical gene' is a gene,
which can be detected with sufficient similarity (p < 10-10) in other
genera.

The alignment between the B.garinii and B.burgdorferi collinear
chromosomes and plasmids was generated using the program stretcher,
which is part of the EMBOSS package (http://www.emboss.org).

The B.garinii sequences were deposited in GenBank with the accession
numbers CP000013 [GenBank] , CP000014 [GenBank] , CP000015 [GenBank]
and AY722917 [GenBank] to AY722953 [GenBank] . The B.garinii genome
data as well as the results of the comparative analysis of B.garinii
with B.burgdorferi are also available from the Spirochetes Genome
Browser at http://sgb.imb-jena.de/. The browser is based on our genome
annotation and analysis system GenColors (manuscript in preparation).

RESULTS
The DNA for the libraries was obtained from the total DNA content of
B.garinii strain PBi. Thus, besides the chromosome, the plasmids of
this strain should also be represented at least in part in the shotgun
data. All sequences from the whole-genome library were binned according
to their similarities to the B.burgdorferi genome parts (i.e.
chromosome and plasmids): 37.8% of all B.garinii reads were derived
from plasmids (Table 1). This value is comparable to that of the
plasmid fraction of B.burgdorferi (40%).

View this table:

   Table 1. Comparison of the B.garinii low redundancy WGS assembly to
the B.burgdorferi sequence

Altogether the assembly comprises 1.227 Mb of B.garinii sequence. Three
contigs completely cover the counterparts of the corresponding
B.burgdorferii chromosome and plasmids lp54 and cp26. Additional 37
contigs >2 kb amounting to 239 kb were obtained for the remaining
plasmid fraction of B.garinii (Figure 1). Clear-cut and error-free
assignment of these plasmid contigs to defined B.burgdorferi plasmids
was not possible, further underlining the variability of the plasmid
complement in Borrelia species. The whole chromosome and two plasmids
(lp54,A and cp26, B) are completely represented in B.garinii. Eight
plasmids of B.burgdorferi are highly similar (cp32: L, M, N, O, P, R,
S; lp56: Q). These seem to be also completely represented by B.garinii
plasmid contigs although neither an exact assignment of individual
contigs to a specific B.burgdorferi plasmid nor the calculation of
their copy number is possible. The remaining plasmid contigs of
B.garinii show also similarities to B.burgdorferi plasmids, but some
regions are either unique to B.burgdorferi or have low DNA
similarities. In four contigs, we observed similarities to different
plasmids of B.burgdorferi indicating breakage/fusion points between
different plasmids.

View larger version (23K):

   Figure 1. View of the B.burgdorferi genome indicating similarities
to sequences of the B.garinii genome. The calculation of the
similarities was done using BLAST. Threshold for identity was 75% on a
length of 40 bases. Portions of the B.burgdorferi genome with matches
are depicted in red, unmatched in blue. The complex consisting of eight
nearly identical plasmids is drawn in yellow. Here, no exact
orthologous regions could be defined since sequences as well as CDSs
are redundant. The scale for the plasmids is 10x magnified compared to
the chromosome.

Chromosome

In our low redundancy project, the average coverage of the chromosome
assembly is 3.38 (Table 1). The initial assembly of the B.garinii
chromosome had 260 gaps (comprising sequencing and clone gaps). After
applying gap closure procedures, we obtained one contig covering almost
(99.5%) the complete linear B.burgdorferi chromosome. Despite the low
redundancy of the sequence reads >80% of the chromosome is endowed with
a error frequency of <10-4. The overall expected error rate is 0.26%
(Supplementary Figure S1). The comparative analysis shows an unbroken
collinearity between the B.burgdorferi and B.garinii chromosomes. The
only parts of the B.burgdorferi chromosome with no counterpart in
B.garinii reside in both telomeric regions with a size of 168 and 8458
bp, respectively. Since ends of linear chromosomes are not clonable
without further manipulation, the missing bases on the left end are
probably due to a cloning bias. It was previously shown that the right
end of the chromosome in B.burgdorferi exhibits length variations in
defined steps in different strains (33). The shorter right end of the
B.garinii chromosome is comparable to one form of these stepwise
variable lengths of the B.burgdorferi chromosomes.

Substitutions, insertions and deletions

Base substitutions are a measure for evolutionary distance between
organisms. The overall identity of the B.garinii with the B.burgdorferi
chromosome is 92.7%. A calculation based on the shared CDS on amino
acid level gives the same result indicating an equal distribution of
substitutions over the chromosome irrespective of information content.
The decrease of similarity between the two chromosomes below 80% in
three regions around the origin of replication as can be seen in Figure
2 is apparently caused by larger insertions and deletions (indels;
Figure 2, number 4-6). In total, we found 66 482 single base
substitutions (Table 2). Transitions and transversions are almost
equally distributed in the genic and intergenic regions.

View larger version (23K):

   Figure 2. A sketch of the B.garinii chromosome compared to the
collinear B.burgdorferi counterpart. Base identity of B.garinii versus
B.burgdorferi is shown as green line, GC content as light blue line
(both left scale) and GC skew as purple line. All values were
calculated using a sliding window of 10 kb with a step width of 1 kb.
Positions of all indels (deletions-negative peaks,
insertions-positive peaks) in the alignment of the two analysed
chromosomes are shown as black bars (right scale). Indels with a
distance <10 bp to each other were defined as one single indel. All
larger indels are indexed with numbers in red, exact positions are
given in Supplementary Table S1. A cluster of five insertions appears
in this figure as one peak (number 4), in a similar manner two
deletions are located near position 540 000 (number 5 together with an
insertion). The deletion of 8458 bases at the telomeric end is not
shown.

View this table:

   Table 2. Frequency of single base substitutions in the collinear
genomic elements of B.burgdorferi and B.garinii

Besides the shorter right end of the chromosome, we found eight
insertions and six deletions with a size >100 bp (Figure 2, numbers
1-8; Supplementary Table S1) in the B.garinii chromosome as major
structural differences relative to the chromosome of B.burgdorferi.
The largest observed insertion with a size of 1878 bp is caused by a
duplication of a region containing the bmpA gene and part of the bmpB
gene (see below) resulting in a tandem repeat of these genes (Figure 2,
number 3). A series of five insertions is separated by short
orthologous sequences of at most 470 bases (Figure 2, number 4;
Supplementary Table S1). This cluster of insertions is located in a
region containing two tRNA genes (tRNA-Ile-1, tRNA-Ala-1) and expands
only intergenic regions.

Indel region 5 consists of an insertion of 538 bases (Figure 2, peak 3)
followed by two deletions of 211 and 498 bases, respectively, which are
separated by 133 bases. In this indel region 5 resides the gene
encoding inositol monophosphatase (BB0524) in B.burgdorferi. This gene
is only partly represented (59 of 284 amino acids) in B.garinii. The
eighth insertion (Figure 2, number 7) is located in an intergenic
region. According to the observed indel regions, two 'hotspots' for
rearrangements on the Borrelia chromosome could be defined: indel
region 4 and indel region 5. Most interestingly, these two regions are
located in close vicinity to the origin of replication of the
chromosome at position 475 kb.

All deletions >100 bases including the missing chromosomal ends
comprise 10 448 bases, all insertions 4099 bases. Thus, the B.garinii
chromosome is by 0.7% shorter than that of B.burgdorferi (Table 2).

Genes

A comparative annotation of the B.garinii chromosome was performed
using the previously published B.burgdorferi gene prediction and
annotation (GenBank NC_001318 [GenBank] .1) (16). In parallel, we used
GeneMarkS for ab initio gene predictions. This program was not able to
detect 36 of the original gene predictions on the B.burgdorferi
chromosome (Supplementary Table S3). Two of these non-verified coding
sequences are fused to neighbouring coding sequences in B.garinii
(BB0410, BB0510). The failure of GeneMarkS to identify BB0412 is
possibly a false negative result, as the program predicted the ortholog
in B.garinii. Additional ten potential genes of the remaining 814 genes
annotated on the chromosome of B.burgdorferi are not predicted on the
chromosome of B.garinii (Supplementary Table S4). Interestingly, these
genes are annotated in B.burgdorferi only as predicted coding region
without any other supportive evidence like similarities to other genes.
Twelve predicted B.burgdorferi genes are fused in B.garinii (BB0078 +
BB0079, BB0356 + BB0357, BB0410 + BB0411, BB0510 + BB0511, BB0521 +
BB0522, BB0710 + BB0711). Eight annotated genes show extensive length
divergence and overlap only partly their B.garinii counterpart, seven
of which are altered due to differing open reading frames (ORFs) on an
otherwise orthologous genomic sequence (BB0475, BB0524, BB0532, BB0546,
BB0591, BB0749, BB0758). The lmp1 gene (BG0212) is affected by the
largest deletion in B.garinii (Figure 2, number 1) and thereby
shortened by 648 bases at the 5' end. In summary, 786 GenBank annotated
genes of B.burgdorferi are supported by GeneMarkS predictions in both
Borrelia species and thus most likely represent the orthologous set of
chromosomal genes. The length of 452 orthologs is unaltered, 255 genes
are longer in B.burgdorferi and 79 genes are longer in B.garinii, but
since these length differences are only small, the core of the deduced
amino acid sequence is not affected.

BB0086 is split in B.garinii. Two genes are affected by a large
duplication in B.garinii leading to a second copy of bmpA (BB0382) and
a partial copy of bmpB (BB0383; Figure 2, number 3). Due to nonsense
mutations, this partial copy is represented in the predicted gene set
by four small ORFs.

In addition to the 807 predicted B.garinii genes with homologous
sequences (including split, fused and other altered gene structures) in
annotated CDS of B.burgdorferi, GeneMarkS predicts 33 further genes.
These genes are comparably small (<60 amino acids) and most likely
represent false positive predictions. This is further underlined by the
fact that four of these potential genes lie within rRNA and tRNA gene
regions and additional four predictions are apparently derived from the
truncated copy of bmpB. The additional 39 genes on the B.burgdorferi
chromosome, which are predicted by GeneMarkS, may also be false
positive predictions.

On the DNA level, no predicted protein-coding gene of B.garinii is
identical to its ortholog in B.burgdorferi whereas 20 tRNA genes (out
of a total of 33 tRNA genes) are identical to their orthologous
counterparts. Interestingly, the mutations seem to affect tRNA genes
not randomly, since most sequence changes occur in non-unique tRNA
genes (11 of 13). All four copies of tRNA-Leu, all three copies of
tRNA-Ser, two of the three tRNA-Arg copies, and the second copy of
tRNA-Lys and tRNA-Thr, respectively, are mutated.

Due to the high similarity of the chromosomes, the statistics of the
codon usage shows only a slight difference between the two species. For
example, in B.burgdorferi a higher preference for TTG as a start codon
than in B.garinii predicted genes could be observed (Supplementary
Table S2).

On the protein level, only 11 of all B.garinii genes are not altered in
comparison to B.burgdorferi. This includes the ribosomal proteins rpsU,
rpsL, rpmG, rpsJ, rpsS, rpmF, a putative subunit K of an ATPase, the
flagellar motor switch protein fliG-2, the phosphocarrier protein
ptsH-2, and the chemotaxis-related proteins cheX and cheY-3.

Additional 25 genes are affected by conservative exchanges with amino
acids having the same chemical properties, thus increasing the number
of highly conserved proteins to 38; not surprisingly 18 genes of this
expanded group code for ribosomal proteins.

As an indication for positive selection, 94 (11.7%) of all orthologous
genes and genes with similar sequences contain more non-synonymous than
synonymous exchanges. Of these, 61 have no functional assignments.
Interestingly, a higher than average proportion of the deduced amino
acid sequences is predicted to contain transmembrane domains (39%
compared to 26% for all proteins, Supplementary Table S8). The
remaining 33 predicted genes are listed in Table 3. Many of these
proteins seem to be located, according to their function, on the
surface of the cell.

View this table:

   Table 3. Proteins with ascribed function with more non-synonymous
(Ka) than synonymous (Ks) codons

Plasmids

Different strains of the same Borrelia species can carry different sets
of plasmids. The differing plasmid repertoire of the cells can be
partly a function of the living conditions (34). Additionally, strains
can loose parts of their equipment due to a lack of selection pressure
(35). Since the primary assembly of the chromosomal reads without
additional gap closure sequences resulted in 91% coverage of the
chromosome, we may conclude that also the plasmids are represented in
the same range of coverage. Using the whole-genome shotgun data, it was
possible to assemble two individual plasmids of the B.garinii PBi
strain completely (Table 1). These two plasmids are highly similar and
collinear to the linear plasmid lp54 and the circular plasmid cp26 of
B.burgdorferi B31. The nearly two times higher coverage of one of these
plasmids (lp54) compared to that of the chromosome indicates that it
should be present in about two copies per cell. Compared to the
chromosome, we find an equal number of base substitutions (8.9%) on the
cp26 plasmid. Interestingly, with 15% substitutions are twice as
frequent on plasmid lp54. Most remarkably, the transition:transversion
ratio on lp54 is 3:2, whereas that of the chromosome as well as that of
cp26 is approximately 4:1 (Table 2). The coding capacity of both
plasmids is comparable to their counterparts in B.burgdorferi. Only
three B.garinii lp54 genes predicted by GenMark are orphans, whereas
the majority of predicted genes (49 of 74) have orthologs in the
B.burgdorferi plasmid: 22 of the predicted genes match as pairs to
different parts of 11 B.burgdorferi genes indicating nonsense mutations
leading to split CDSs in B.garinii. On the other hand, 14 predicted
B.burgdorferi genes have no counterparts on the B.garinii plasmid
(Supplementary Table S5). None of these genes has an ascribed function.
Interestingly, the B.burgdorferi lp54 gene family (BBA68 to BBA73)
appears to be almost completely conserved in B.garinii, only BBA72
being split into two predicted genes. The analysis of the coding
capacity of cp26 showed that all 26 predicted B.garinii genes have
orthologs in B.burgdorferi, only three (BBB15, BBB20, BBB21) of the
B.burgdorferi predicted genes are orphans (Supplementary Table S5).
Interestingly, two cp26 encoded genes are subjected to a rapid positive
selection: ospC and BBB08. OspC is well characterized as outer surface
protein, whereas BBB08 so far has no assigned function. On the other
hand, only 17 of the 55 lp54 encoded proteins may be subjected to a
neutral evolution or purifying selection.

The assignment of clusters of orthologous groups (COG) (36) to the
predicted proteins is clearly different between the chromosome and the
plasmids. Whereas 81.6% of the chromosome-encoded orthologous proteins
can be assigned to a COG, only 53.9% (cp26) and 26.5% (lp54) can be
categorized this way (Supplementary Table S6).

All other B.garinii plasmids are represented in our assembly as 37
contigs >2 kb comprising 239 kb. We here refer to these plasmid parts
as variable plasmid segments (VPS). As it is known from the assembly of
the B.burgdorferi plasmids, there are redundant segments distributed
over several plasmids (37,38). The same holds true for the B.garinii
VPS. Some redundant regions containing polymorphisms could separately
be assembled. Yet, the read coverage of some contigs is higher than
that of the chromosome and cp26. Thus, it is very likely that these
portions of the VPS represent paralogous sequences. Therefore, they
cannot be assembled properly into individual plasmids. Accordingly, due
to this non-unique nature of many segments in the plasmids, a clear 1:1
assignment to defined plasmids of B.burgdorferi is not possible.

A GeneMarkS gene prediction revealed 338 complete and truncated
potential protein-coding genes on the VPS: 284 of these predicted genes
have matches to predicted genes in the B.burgdorferi genome on protein
level. Many, mainly small genes (117) show partial matches, but 167
predicted genes exhibit similar lengths in both genomes. One of these
genes is a true ortholog to BB0844, which is encoded on the chromosome
in B.burgdorferi. All other predicted genes are related to
plasmid-encoded genes.

To get an overview of our B.garinii VPS assembly, we performed a BLAST
search on nucleotide level of all plasmid-derived contigs against a
database of B.burgdorferi plasmids. This BLAST search revealed that 70%
(167 kb) of the VPS are similar enough to B.burgdorferi plasmid
sections to be detected (Figure 1). The remaining sequences (73 kb)
have no detectable similarity to B.burgdorferi plasmids on the DNA
level. Yet, if we search for similarities on protein level, we find
matches (40-70% identity on amino acid level) for all contigs to
putative B.burgdorferi plasmid-encoded proteins. The contig with the
lowest similarity to B.burgdorferi sequences is contig AY722928
[GenBank] , which encodes a vls locus involved in antigenic variation
in the mammalian host (39). This locus is located on lp28-1 in
B.burgdorferi. Thus, all VPS sequences seem to be represented in
B.burgdorferi plasmids.

We then asked, which part of the coding capacity of the B.burgdorferi
plasmids is present in B.garinii. Since small predicted genes are often
false positives, for a reliable comparison of the gene sets, we took
into account only B.burgdorferi plasmid-derived proteins >100 amino
acids, and searched for their counterparts in the whole B.garinii
shotgun data. Only one protein each from plasmids lp5 (T), lp54 (Q),
lp21 (U), cp32-3 (S), lp25 (E), lp28-1 (F), lp28-2 (G), lp28-3 (H) and
lp28-4 (I) had no counterpart. The failure to detect these proteins in
the B.garinii genome could be due to missing shotgun data.
Interestingly, from plasmids lp38 (J) 15 of 21 and from plasmid lp36
(K) 9 of 24 proteins were not represented in the shotgun data
(Supplementary Table S7). A more detailed inspection revealed that the
predicted protein-coding regions from these two plasmids that have
matches to the shotgun data belong to protein families. Members of
these protein families are encoded also on different plasmids. These
results taken together indicate that B.garinii PBi lacks the
counterparts of plasmids lp38 and lp36.

Since the copy number of plasmid segments can affect the phenotype of
Borreliae (40), we also analysed the data in this respect. According to
the BLAST hits against B.burgdorferi proteins >100 amino acids plasmids
lp28-1 (F), lp28-3 (H), lp28-4 (I), lp17 (D), lp25 (E) and lp5 (T) are
present in one copy per cell. Plasmid lp28-2 is represented with two
independent segments in our assembly. Thus, it should exist as two
slightly different copies in B.garinii. For the proteins from the
highly redundant cp32 and cp56 plasmids, we observed between three and
four copies each. Furthermore, since parts of these segments are
identical, the assembly resulted in parts of these segments in a higher
coverage than average. We thus estimate that this plasmid group is at
least present in five copies. The plasmid cp9 encodes similar proteins
as the cp32 plasmids, albeit with much lower similarities. Thus, we are
not able to determine whether a counterpart of this particular plasmid
belongs to the B.garinii genome.

DISCUSSION

Closely related pathogenic species can cause different symptoms or even
a different disease based on unique species-specific features. To
reveal the molecular basis of such differences, one can examine the
genomic repertoire of two closely related species. In cases where
previous studies revealed not only a high similarity on DNA level but
also almost complete collinearity of the chromosomes or large segments
thereof, a direct comparative analysis without a completely finished
genome is feasible (41,42). The limitation of this method lies in the
inability to resolve highly rearranged and repetitive structures of the
genome. Here, we report on an in-depth exploration of the B.garinii
genome in comparison to that of B.burgdorferi. The analysis revealed a
complete collinearity of the chromosome as well as of two plasmids in
the compared species. The other genome parts seem to be subjected to
rapid sequence changes as well as rearrangements and duplications.
Thus, Borreliae genomes seem to consist of a remarkably invariant part
mainly responsible for survival in ticks and a fluctuating part
responsible for pathogenicity and disease symptoms in humans.

Borrelia core gene repertoire annotation on the invariant collinear
genome fraction

An ab initio prediction of genes is flawed by the uncertainty as to
what constitutes a real gene in a given organism (43). In recent years,
it was successfully shown that a comparative genomics approach could
improve if not largely replace an annotation from scratch (44,45). If
the relationship between the two compared species is close enough, most
if not all genes should have an ortholog in the sister species. Thus,
the use of orthology information is the best approach to discern
between true genes and false positive predictions or species-specific
adaptations. On the stable fraction of the genome (chromosome and
plasmids lp54 and cp26), we could easily define, which of the predicted
genes in both organisms are orthologous gene pairs. Some of the
orthologous pairs found may not be true genes especially if they are
short. But the high conservation would suggest at least a regulatory
function of these chromosomal regions. In total, we found 861
orthologous gene pairs compared to 955 (B.burgdorferi) and 932
(B.garinii) predicted genes on these conserved genomic elements. Since
the other plasmids seem to be dispensable for viability, this set of
orthologous protein-coding gene pairs is very likely the basic
repertoire of Borrelia burgdorferi sensu lato species. This is
supported by the fact that for example the proteins OspA, OspB and
OspC, which seem to be required for survival in the tick midguts (46),
are encoded on the two conserved plasmids.

COG categories show interspecies relationships, i.e. only proteins more
common than for one genus can be categorized this way (36). The
fraction of categorizable proteins decreases from the chromosome to
cp26 and then to lp54 considerably, whereas the fraction of positively
selected genes increases. Recently, it was shown that B.burgdorferi is
not able to live without cp26 (47). Our analysis further supports the
view that cp26 is an essential genome part of Borrelia species. Both
the presence of two members of a family of genes (ospA and ospB) known
to be needed in tick midguts and the conserved collinearity of the
plasmid leads us to the conclusion that lp54 very likely plays also a
pivotal role in survival in ticks. Since most of the encoded proteins
are subjected to positive selection, we hypothesize that lp54 is a
major player in host response evasion.

Selection pressure
The analysis of the ratio of non-synonymous to synonymous substitutions
shows that the overwhelming majority of functionally described proteins
of the chromosome (475/513; 92.6%) are under neutral evolution or
purifying selection. The remaining 33 proteins (Table 3) show positive
selection to different degrees. As expected, we find many
surface-associated proteins in this list, which may be involved in the
escape from the host response. On the other hand, of the 295 proteins
described only as predicted or hypothetical, 56 (19%) seem to be under
positive selection. This is a larger fraction than that in the subset
with known function. Many of the proteins with functional assignment as
well as the hypothetical proteins seem to be located at the surface of
the cell and presumably generate a strain variability to fool the
immune response from ticks as well as from humans. Thus, this subset of
hypothetical proteins would be a rewarding target for further
functional studies.

Variable plasmid complement of B.garinii
Only an isolation and analysis of each single plasmid would allow the
resolution of the entire plasmid fraction of B.garinii. But since the
whole-genome shotgun data should represent most parts of the plasmids
(see Results), we are able to calculate similarities between contigs
and plasmids to uncover the relationship of B.garinii to B.burgdorferi
plasmids, and to reveal differences in plasmid content between the two
species.

The noncollinear plasmids are not only rearranged but also the encoded
proteins are not as conserved as in the collinear genome parts. Yet,
two-thirds of the B.garinii VPS sequences are similar enough on DNA
level to match B.burgdorferi counterparts. For the remaining sequences,
no DNA similarities could be found. Nevertheless, our ability to detect
similarities on the protein level shows that these plasmid parts are
more likely subject to an accelerated evolution than unique B.garinii
genome constituents: 167 of the 338 predicted genes have counterparts
of similar length in the B.burgdorferi genome. Despite their high
divergence, they may thus be ascribed as proteins with orthologous
functions. BB0844 is encoded on the right-end extension of the
B.burgdorferi chromosome and its ortholog on a plasmid in B.garinii.
This may point to a mechanism, which enables variable Borrelia
chromosome lengths by exchanging parts between chromosome and distinct
plasmid segments.

Based on comparisons of the coding capacity of the VPS with the
B.burgdorferi plasmids, we are able to define, which plasmids are
presumably present in B.garinii and which not. With this analysis, we
could show that two plasmids (lp38, J and lp36, K) are missing from the
B.garinii genome. This is especially important since lp38 encodes a
member of the osp family of proteins (ospD), a protein family widely
studied for its role in pathogenicity and survival in the hosts.

It was shown that interplasmidal duplications and rearrangements are
able to change the virulence phenotype of Borrelia species (40). Thus,
it is of high value to know, which plasmids or segments are represented
more than once in the genome. Generally, duplication and
diversification events seem to affect the same plasmids in the two
species. We also see an amplification of the cp32 plasmid sequences,
although there may be a few copies less in B.garinii than in
B.burgdorferii. Most other plasmid sequences are represented only as
one copy in each species. Yet, in B.garinii plasmid lp28-2 sequences
underwent also duplication and diversification. Thus, while the main
difference in the protein family sets lies in the presence or absence
of plasmids lp36 and lp38, additional diversity is achieved not only by
mutation of single genes but also by a duplication of lp28-2, and
possibly modification of the copy number of cp32 plasmid sequences.

Evolution and species definition

In previous studies (48-50), a large genetic distance between and
within Borrelia species was observed on the basis of highly variable
genes and genomic regions. Most of the genes examined are located on
the plasmids, which are far less conserved than the chromosome. In
contrast, we found a strong conservation of similarity and collinearity
between B.garinii and B.burgdorferi not only of the chromosome but also
of two plasmids. Interestingly, the amino acid identity of
chromosomally encoded proteins is not higher than the conservation of
the whole chromosome on DNA level. Thus, despite positive selection
observed for specific proteins (Table 3), the chromosome on the whole
is subjected to a neutral evolution.

Since plasmid repertoire variability is observed also in closely
related strains causing similar disease patterns, the species
definition is based only on the chromosome. Proteins encoded on the
chromosome may also slightly influence the disease pattern. As
discussed before, the survival in ticks may be mediated by the two
collinear plasmids. Thus, pathogenicity in vertebrate hosts could be
mainly dependent on the VPS. Future work has to determine, which
plasmids or plasmid parts are lost during loss of pathogenicity. Since
both Borrelia species have amplified mainly the same plasmids or
segments thereof, it is conceivable that it is not enough to keep one
member of each plasmid-encoded protein for maintenance of
pathogenicity. Rather a larger number of paralogous proteins encoded by
redundant plasmids could be required to successfully infect
vertebrates.

Possibly the genes under positive selection are also causative for the
symptom differences of the various Borrelia species. Thus, both the
variable plasmid part of the genome and the positively selected genes
represent prime targets for further functional studies.

    SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online.

    REFERENCES

Wang,G., van Dam,A.P., Schwartz,I. and Dankert,J. ( (1999) ) Molecular
typing of Borrelia burgdorferi sensu lato: taxonomic, epidemiological,
and clinical implications. Clin. Microbiol. Rev., , 12, ,
633-653.[Abstract/Free Full Text]

Marti Ras,N., Postic,D., Foretz,M. and Baranton,G. ( (1997) ) Borrelia
burgdorferi sensu stricto, a bacterial species 'made in the
U.S.A.'? Int. J. Syst. Bacteriol., , 47, , 1112-1117.[Abstract/Free
Full Text]

Wilske,B., Busch,U., Eiffert,H., Fingerle,V., Pfister,H.W., Rossler,D.
and Preac-Mursic,V. ( (1996) ) Diversity of OspA and OspC among
cerebrospinal fluid isolates of Borrelia burgdorferi sensu lato from
patients with neuroborreliosis in Germany. Med. Microbiol. Immunol.
(Berl.), , 184, , 195-201.[ISI][Medline]

Saint Girons,I., Gern,L., Gray,J.S., Guy,E.C., Korenberg,E.,
Nuttall,P.A., Rijpkema,S.G., Schonberg,A., Stanek,G. and Postic,D. (
(1998) ) Identification of Borrelia burgdorferi sensu lato species in
Europe. Zentralbl. Bakteriol., , 287, , 190-195.[ISI][Medline]

Stevenson,B. and Miller,J.C. ( (2003) ) Intra- and interbacterial
genetic exchange of Lyme disease spirochete erp genes generates
sequence identity amidst diversity. J. Mol. Evol., , 57, ,
309-324.[ISI]

Purser,J.E., Lawrenz,M.B., Caimano,M.J., Howell,J.K., Radolf,J.D. and
Norris,S.J. ( (2003) ) A plasmid-encoded nicotinamidase (PncA) is
essential for infectivity of Borrelia burgdorferi in a mammalian host.
Mol. Microbiol., , 48, , 753-764.[ISI][Medline]

Lagal,V., Postic,D. and Baranton,G. ( (2002) ) Molecular diversity of
the ospC gene in Borrelia. Impact on phylogeny, epidemiology and
pathology. Wien. Klin. Wochenschr., , 114, , 562-567.[ISI][Medline]

Canica,M.M., Nato,F., du Merle,L., Mazie,J.C., Baranton,G. and
Postic,D. ( (1993) ) Monoclonal antibodies for identification of
Borrelia afzelii sp. nov. associated with late cutaneous manifestations
of Lyme borreliosis. Scand. J. Infect. Dis., , 25, ,
441-448.[ISI][Medline]

Lunemann,J.D., Zarmas,S., Priem,S., Franz,J., Zschenderlein,R.,
Aberer,E., Klein,R., Schouls,L., Burmester,G.R. and Krause,A. ( (2001)
) Rapid typing of Borrelia burgdorferi sensu lato species in specimens
from patients with different manifestations of Lyme borreliosis. J.
Clin. Microbiol., , 39, , 1130-1133.[Abstract/Free Full Text]

Eiffert,H., Karsten,A., Thomssen,R. and Christen,H.J. ( (1998) )
Characterization of Borrelia burgdorferi strains in Lyme arthritis.
Scand. J. Infect. Dis., , 30, , 265-268.[CrossRef][ISI][Medline]

Vasiliu,V., Herzer,P., Rossler,D., Lehnert,G. and Wilske,B. ( (1998) )
Heterogeneity of Borrelia burgdorferi sensu lato demonstrated by an
ospA-type-specific PCR in synovial fluid from patients with Lyme
arthritis. Med. Microbiol. Immunol. (Berl.), , 187, ,
97-102.[CrossRef][ISI][Medline]

Xu,Y. and Johnson,R.C. ( (1995) ) Analysis and comparison of plasmid
profiles of Borrelia burgdorferi sensu lato strains. J. Clin.
Microbiol., , 33, , 2679-2685.[Abstract]

Casjens,S., Delange,M., Ley,H.L.,III, Rosa,P. and Huang,W.M. ( (1995) )
Linear chromosomes of Lyme disease agent spirochetes: genetic diversity
and conservation of gene order. J. Bacteriol., , 177, ,
2769-2780.[Abstract]

Schwan,T.G., Burgdorfer,W. and Garon,C.F. ( (1988) ) Changes in
infectivity and plasmid profile of the Lyme disease spirochete,
Borrelia burgdorferi, as a result of in vitro cultivation. Infect.
Immun., , 56, , 1831-1836.[ISI][Medline]

Busch,U., Will,G., Hizo-Teufel,C., Wilske,B. and Preac-Mursic,V. (
(1997) ) Long-term in vitro cultivation of Borrelia burgdorferi sensu
lato strains: influence on plasmid patterns, genome stability and
expression of proteins. Res. Microbiol., , 148, ,
109-118.[CrossRef][ISI][Medline]

Fraser,C.M., Casjens,S., Huang,W.M., Sutton,G.G., Clayton,R.,
Lathigra,R., White,O., Ketchum,K.A., Dodson,R., Hickey,E.K. et al. (
(1997) ) Genomic sequence of a Lyme disease spirochaete, Borrelia
burgdorferi. Nature, , 390, , 580-586.[CrossRef][ISI][Medline]

Casjens,S., Palmer,N., van Vugt,R., Huang,W.M., Stevenson,B., Rosa,P.,
Lathigra,R., Sutton,G., Peterson,J., Dodson,R.J. et al. ( (2000) ) A
bacterial genome in flux: the twelve linear and nine circular
extrachromosomal DNAs in an infectious isolate of the Lyme disease
spirochete Borrelia burgdorferi. Mol. Microbiol., , 35, ,
490-516.[CrossRef][ISI][Medline]

Xu,Y., Kodner,C., Coleman,L. and Johnson,R.C. ( (1996) ) Correlation of
plasmids with infectivity of Borrelia burgdorferi sensu stricto type
strain B31. Infect. Immun., , 64, , 3870-3876.[Abstract]

Elias,A.F., Stewart,P.E., Grimm,D., Caimano,M.J., Eggers,C.H.,
Tilly,K., Bono,J.L., Akins,D.R., Radolf,J.D., Schwan,T.G. et al. (
(2002) ) Clonal polymorphism of Borrelia burgdorferi strain B31 MI:
implications for mutagenesis in an infectious strain background.
Infect. Immun., , 70, , 2139-2150.[Abstract/Free Full Text]

Ren,S.X., Fu,G., Jiang,X.G., Zeng,R., Miao,Y.G., Xu,H., Zhang,Y.X.,
Xiong,H., Lu,G., Lu,L.F. et al. ( (2003) ) Unique physiological and
pathogenic features of Leptospira interrogans revealed by whole-genome
sequencing. Nature, , 422, , 888-893.[CrossRef][ISI][Medline]

Seshadri,R., Myers,G.S., Tettelin,H., Eisen,J.A., Heidelberg,J.F.,
Dodson,R.J., Davidsen,T.M., DeBoy,R.T., Fouts,D.E., Haft,D.H. et al. (
(2004) ) Comparison of the genome of the oral pathogen Treponema
denticola with other spirochete genomes. Proc. Natl Acad. Sci. USA, ,
101, , 5646-5651. Epub 2004 Apr 5642.[Abstract/Free Full Text]

Fraser,C.M., Norris,S.J., Weinstock,G.M., White,O., Sutton,G.G.,
Dodson,R., Gwinn,M., Hickey,E.K., Clayton,R., Ketchum,K.A. et al. (
(1998) ) Complete genome sequence of Treponema pallidum, the syphilis
spirochete. Science, , 281, , 375-388.[Abstract/Free Full Text]

Zhong,J. and Barbour,A.G. ( (2004) ) Cross-species hybridization of a
Borrelia burgdorferi DNA array reveals infection- and
culture-associated genes of the unsequenced genome of the relapsing
fever agent Borrelia hermsii. Mol. Microbiol., , 51, ,
729-748.[ISI][Medline]

Kellis,M., Patterson,N., Endrizzi,M., Birren,B. and Lander,E.S. (
(2003) ) Sequencing and comparison of yeast species to identify genes
and regulatory elements. Nature, , 423, ,
241-254.[CrossRef][ISI][Medline]

Hardison,R.C. ( (2003) ) Comparative genomics. PLoS Biol., , 1, , E58.
Epub 2003 Nov 2017.[Medline]

Ojaimi,C., Davidson,B.E., Saint Girons,I. and Old,I.G. ( (1994) )
Conservation of gene arrangement and an unusual organization of rRNA
genes in the linear chromosomes of the Lyme disease spirochaetes
Borrelia burgdorferi, B.garinii and B.afzelii. Microbiology, , 140, ,
2931-2940.[ISI][Medline]

Wilske,B., Preac-Mursic,V., Gobel,U.B., Graf,B., Jauris,S.,
Soutschek,E., Schwab,E. and Zumstein,G. ( (1993) ) An OspA serotyping
system for Borrelia burgdorferi based on reactivity with monoclonal
antibodies and OspA sequence analysis. J. Clin. Microbiol., , 31, ,
340-350.[Abstract]

Preac-Mursic,V., Wilske,B. and Reinhardt,S. ( (1991) ) Culture of
Borrelia burgdorferi on six solid media. Eur. J. Clin. Microbiol.
Infect. Dis., , 10, , 1076-1079.[ISI][Medline]

Roe,B.A. ( (2004) ) Shotgun library construction for DNA sequencing.
Methods Mol. Biol., , 255, , 171-187.[Medline]

Altschul,S.F., Madden,T.L., Schaffer,A.A., Zhang,J., Zhang,Z.,
Miller,W. and Lipman,D.J. ( (1997) ) Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids Res., ,
25, , 3389-3402.[Abstract/Free Full Text]

Bonfield,J.K., Smith,K. and Staden,R. ( (1995) ) A new DNA sequence
assembly program. Nucleic Acids Res., , 23, , 4992-4999.[Abstract]

Besemer,J., Lomsadze,A. and Borodovsky,M. ( (2001) ) GeneMarkS: a
self-training method for prediction of gene starts in microbial
genomes. Implications for finding sequence motifs in regulatory
regions. Nucleic Acids Res., , 29, , 2607-2618.[Abstract/Free Full
Text]

Casjens,S., Murphy,M., DeLange,M., Sampson,L., van Vugt,R. and
Huang,W.M. ( (1997) ) Telomeres of the linear chromosomes of Lyme
disease spirochaetes: nucleotide sequence and possible exchange with
linear plasmid telomeres. Mol. Microbiol., , 26, ,
581-596.[ISI][Medline]

Iyer,R., Kalu,O., Purser,J., Norris,S., Stevenson,B. and Schwartz,I. (
(2003) ) Linear and circular plasmid content in Borrelia burgdorferi
clinical isolates. Infect. Immun., , 71, , 3699-3706.[Abstract/Free
Full Text]

Grimm,D., Elias,A.F., Tilly,K. and Rosa,P.A. ( (2003) ) Plasmid
stability during in vitro propagation of Borrelia burgdorferi assessed
at a clonal level. Infect. Immun., , 71, , 3138-3145.[Abstract/Free
Full Text]

Natale,D.A., Galperin,M.Y., Tatusov,R.L. and Koonin,E.V. ( (2000) )
Using the COG database to improve gene recognition in complete genomes.
Genetica, , 108, , 9-17.[CrossRef][ISI][Medline]

Stevenson,B., Zuckert,W.R. and Akins,D.R. ( (2000) ) Repetition,
conservation, and variation: the multiple cp32 plasmids of Borrelia
species. J. Mol. Microbiol. Biotechnol., , 2, ,
411-422.[ISI][Medline]

Zuckert,W.R. and Meyer,J. ( (1996) ) Circular and linear plasmids of
Lyme disease spirochetes have extensive homology: characterization of a
repeated DNA element. J. Bacteriol., , 178, , 2287-2298.[Abstract]

Zhang,J.R., Hardham,J.M., Barbour,A.G. and Norris,S.J. ( (1997) )
Antigenic variation in Lyme disease borreliae by promiscuous
recombination of VMP-like sequence cassettes. Cell, , 89, ,
275-285.[ISI][Medline]

Penningon,P.M., Cadavid,D., Bunikis,J., Norris,S.J. and Barbour,A.G. (
(1999) ) Extensive interplasmidic duplications change the virulence
phenotype of the relapsing fever agent Borrelia turicatae. Mol.
Microbiol., , 34, , 1120-1132.[CrossRef][ISI][Medline]

Kirkness,E.F., Bafna,V., Halpern,A.L., Levy,S., Remington,K.,
Rusch,D.B., Delcher,A.L., Pop,M., Wang,W., Fraser,C.M. et al. ( (2003)
) The dog genome: survey sequencing and comparative analysis. Science,
, 301, , 1898-1903.[Abstract/Free Full Text]

Anzai,T., Shiina,T., Kimura,N., Yanagiya,K., Kohara,S., Shigenari,A.,
Yamagata,T., Kulski,J.K., Naruse,T.K., Fujimori,Y. et al. ( (2003) )
Comparative sequencing of human and chimpanzee MHC class I regions
unveils insertions/deletions as the major path to genomic divergence.
Proc. Natl Acad. Sci. USA, , 100, , 7708-7713. Epub 2003 Jun
7710.[Abstract/Free Full Text]

Aggarwal,G. and Ramaswamy,R. ( (2002) ) Ab initio gene identification:
prokaryote genome annotation with GeneScan and GLIMMER. J. Biosci., ,
27, , 7-14.[ISI][Medline]

Nowrousian,M., Wurtz,C., Poggeler,S. and Kuck,U. ( (2004) ) Comparative
sequence analysis of Sordaria macrospora and Neurospora crassa as a
means to improve genome annotation. Fungal Genet. Biol., , 41, ,
285-292.[CrossRef][ISI][Medline]

Dubchak,I. and Frazer,K. ( (2003) ) Multi-species sequence comparison:
the next frontier in genome annotation. Genome Biol., , 4, , 122. Epub
2003 Nov 2027.[CrossRef][Medline]

Yang,X.F., Pal,U., Alani,S.M., Fikrig,E. and Norgard,M.V. ( (2004) )
Essential role for OspA/B in the life cycle of the Lyme disease
spirochete. J. Exp. Med., , 199, , 641-648. Epub 2004 Feb
2023.[Abstract/Free Full Text]

Byram,R., Stewart,P.E. and Rosa,P. ( (2004) ) The essential nature of
the ubiquitous 26-kilobase circular replicon of Borrelia burgdorferi.
J. Bacteriol., , 186, , 3561-3569.[Abstract/Free Full Text]

Farlow,J., Postic,D., Smith,K.L., Jay,Z., Baranton,G. and Keim,P. (
(2002) ) Strain typing of Borrelia burgdorferi, Borrelia afzelii, and
Borrelia garinii by using multiple-locus variable-number tandem repeat
analysis. J. Clin. Microbiol., , 40, , 4612-4618.[Abstract/Free Full
Text]

Bunikis,J., Garpmo,U., Tsao,J., Berglund,J., Fish,D. and Barbour,A.G. (
(2004) ) Sequence typing reveals extensive strain diversity of the Lyme
borreliosis agents Borrelia burgdorferi in North America and Borrelia
afzelii in Europe. Microbiology, , 150, ,
1741-1755.[CrossRef][ISI][Medline]

Xu,Y., Bruno,J.F. and Luft,B.J. ( (2003) ) Detection of genetic
diversity in linear plasmids 28-3 and 36 in Borrelia burgdorferi sensu
stricto isolates by subtractive hybridization. Microb. Pathog., , 35, ,
269-278.[CrossRef][ISI][Medline]

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